Differential effects of nitrogenous fertilizers on methane-consuming microbes in rice field and forest soils.

Abstract

The impact of environmental perturbation (e.g., nitrogenous fertilizers) on the dynamics of methane fluxes from soils and wetland systems is poorly understood. Results of fertilizer studies are often contradictory, even within similar ecosystems. In the present study the hypothesis of whether these contradictory results may be explained by the composition of the methane-consuming microbial community and hence whether methanotrophic diversity affects methane fluxes was investigated. To this end, rice field and forest soils were incubated in microcosms and supplemented with different nitrogenous fertilizers and methane concentrations. By labeling the methane with 13C, diversity and function could be coupled by analyses of phospholipid-derived fatty acids (PLFA) extracted from the soils at different time points during incubation. In both rice field and forest soils, the activity as well as the growth rate of methane-consuming bacteria was affected differentially. For type I methanotrophs, fertilizer application stimulated the consumption of methane and the subsequent growth, while type II methanotrophs were generally inhibited. Terminal restriction fragment length polymorphism analyses of the pmoA gene supported the PLFA results. Multivariate analyses of stable-isotope-probing PLFA profiles indicated that in forest and rice field soils, Methylocystis (type II) species were affected by fertilization. The type I methanotrophs active in forest soils (Methylomicrobium/Methylosarcina related) differed from the active species in rice field soils (Methylobacter/Methylomonas related). Our results provide a case example showing that microbial community structure indeed matters, especially when assessing and predicting the impact of environmental change on biodiversity loss and ecosystem functioning.

Dynamics of MOB-specific PLFA in forest soil incubations with different methane concentrations and N amendments. Table shows the growth rates calculated from the increase of PLFA over time. Table SA1 in the supplemental material shows the statistical analyses.

Total 13C incorporation in MOB-specific PLFA in forest soil after 48 days of incubations. Table SA3 in the supplemental material shows the statistical analyses of the effects of methane concentration and N amendment on 13C incorporation.

Total 13C incorporation in MOB-specific PLFA in rice field soil incubations. Incubations were performed using 10,000 ppmv methane and were supplemented with or without ammonium. Table SA4 in the supplemental material shows the statistical analyses.

Multivariate statistical analyses of SIP 13CH4-PLFA profiles of the MOB community in forest soil samples. The inputs of the MDS (A and C) and the cluster analyses (B and D) were PLFA profiles of MOB cultures (expressed as percentage of total PLFA content) and of SIP profiles of soil samples (expressed as percentage of 13C incorporated in separate PLFAs of the total PLFA 13C uptake). The two-dimensional distances between samples in the MDS graph show the relative similarity between samples. Stress values of the MDS plots were 0.12 (A) and 0.09 (C). For the cluster analyses, the profiles were transformed [log(x + 1)] before the Bray-Curtis similarity matrix was established. The clustering was done using the group average linking method.

Multivariate statistical analyses of SIP 13CH4-PLFA profiles of the MOB community in rice field soil samples. The inputs of the MDS (A and C) and the cluster analyses (B and D) were PLFA profiles of MOB cultures (expressed as percentage of total PLFA content) and of SIP profiles of soil samples (expressed as percentage of 13C incorporated in separate PLFAs of the total PLFA 13C uptake). The two-dimensional distances between samples in the MDS graph show the relative similarity between samples. Stress values of the MDS plots were 0.13 (A) and 0.10 (C). For the cluster analyses, the profiles were transformed [log(x + 1)] before the Bray-Curtis similarity matrix was established. The clustering was done using the group average linking method ().

Relative abundance of terminal restriction fragment 245, which is representative of type II MOB, as affected by incubation time, methane concentration, and N amendment in forest and rice field soils. Relative abundance is expressed as the percentage of the total fluorescence recorded per sample. All points represent the means and standard errors for three replicate samples. The statistical evaluation of the data is presented in Table SA5 in the supplemental material.